Nervenzellen sind hochspezialisierte Zellen, die an Synapsen miteinander verbunden sind, was die Übertragung von neuronalen Informationen erlaubt. Die Entwicklung von Synapsen und die Informationsverarbeitung und Gedächtnisbildung bei reifen Synapsen erfordert eine dynamische Umorganisation von neuronalen Netzwerken. Das beinhaltet die Bildung und Entfernung von Synapsen, Umsatz von synaptischen Proteinen und die Veränderung und Anpassung von synaptischer Erregungsübertragung. U. a. Ubiquitinierung, als regulatorische, posttranslationale Modifikation von Proteinen, könnte eine entscheidende Rolle für solche komplexe, synaptische Umorganisationen spielen. Nedd4-1, eine HECT-Typ E3 Ubiquitin Ligase, reguliert und fördert die Entwicklung von Nervenzellfortsätzen durch die Ubiquitinierung von Rap2. Um die Bedeutung von Nedd4-abhänginger Ubiquitinierung im entwickelten Gehirn zu untersuchen, wurden Mausmodelle generiert und analysiert, in denen Nedd4-1 und dessen nächstes Homolog Nedd4-2, speziell in Nervenzellen ausgeschaltet wurde. Ich habe herausgefunden, dass Nedd4-1 und Nedd4-2 wichtige regulatorische Proteine für die neuronale Morphogenese und die synaptische Plastizität, insbesondere die Aufrechterhaltung von LTP, darstellen. Desweiteren habe ich festgestellt, dass Synaptopodin (SYNPO), ein Prolin-reiches, Aktin-assoziiertes Protein, von Nedd4-1 und Nedd4-2 in vitro ubiquitiniert wird. Dieses Ergebnis deutet daraufhin, dass SYNPO in dem Mechanismus eine Rolle spielt, durch den Nedd4-1 und Nedd4-2 LTP aufrechterhalten. Diese Studie wirft ein neues Licht auf die funktionelle Rolle von Nedd4-abhänginger Ubiquitinierung bei höheren Funktionen des Gehirns von Säugetieren sowie der neuronalen Entwicklung.

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The aim of this thesis is to develop a novel technique for the estimation of firing rate dynamics from single-unit recordings of neural pulse trains. This method applies an offline digital filtering technique to extract information transmitted by a neuron in teens of a rate code. While there is increasing evidence that the traditional rate coding cannot account for all the information transmitted by a cell, and that information may also be contained in the precise timing of spikes, the firing rate signal remains the benchmark by which the vast majority of electrophysiological studies relating neural activity to functional behaviour have been interpreted. Nevertheless, there does not seem to be an agreement on a single definition of a rate code let alone a consensus on an optimal estimation method. This study raises significant concerns about the validity of some of the most common methods in systems neuroscience, and proposes a simple yet more robust alternative. This latter is based on the convolution of the spike train with an optimally designed Kaiser window. Using computer-simulated as well as experimental data obtained from single-unit recordings of vestibular canal afferents, the proposed technique is shown to consistently outperform the current methods and even to permit robust estimations under time-varying conditions. These results suggest that estimates acquired with the conventional methods are biased and hence models of neural dynamics based on these latter may not be reliable.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

Mental health of a person depends on the correct functioning of the brain. The brain and the spinal cord contain many types of cells, of which one important type are called the neurons. Neurons are special in the way they connect to each other to form large networks. The chemicals called transmitters are packed at the nerve endings into tiny packets called vesicles and when a signal arrives these vesicles fuse immediately to the attached cell surface and release their contents. The role of the synaptic vesicular transporter proteins is to ensure proper packing of transmitter molecules that can be released upon stimulation. Vesicular packing is an important process. The carrier proteins involved in packing work in coordination to determine the amount and type of transmitters to be packed. Missing a carrier protein from the vesicles might lead to improper packing and inaccurate signaliing. These signaling molecules are known for their implications in many psychiatric and neurological disorders like Alzheimer’s disease, Parkinson’s disease, Schizophrenia, and attention deficit to name just a few.  How a vesicular transporter can affect the modulatory functions of aminergic neurons is the subject of this thesis. This thesis reports on the effects of the loss of a vesicular orphan transporter. Study I demonstrates the localization of this protein to monoaminergic and cholinergic terminals. It reports the effect of the loss of Slc10A4 on vesicular dopamine uptake, synaptic clearance of dopamine and hypersensitivity of animals to dopamine related psychostimulants. Study I also provides evidence for ATP as a possible ligand for SLC10A4 protein. Study II provides data on the clinical relevance of Slc10A4 in playing a protective role against vulnerability to epilepsy. It reports that loss of Slc10A4 renders the animals hypersensitive to cholinergic drugs. Study III provides a closer look at individual cholinergic synapses at neuromuscular junctions in mice lacking Slc10A4. The structural and electrophysiological properties of the NMJ are found compromised because of the loss of this vesicular protein. Taken together, this thesis presents a SV protein’s perspective of viewing at modulation of synaptic transmission.

Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.